Preparation of Transgenic Construct for Pronuclear Injection

Alison Hayward and Aurora Burds Connor, Jan 2007

This protocol is for constructs that will integrate randomly into the mouse genome. To prepare a construct for targeting of a specific mouse locus, see Targeting Construct Linearization. For more information on the differences between these types of mice, please see Intro to Mouse Models.

Before your injection can occur, you will need to obtain approval from the MIT Committee on Animal Care (CAC), a protocol number for your intended experiments using your transgenic mice, and space available in a mouse room before we will inject your DNA to make mice. Forms can be found at the DCM website (MIT Certificate Required). This can take 6-8 weeks, so start early!

Designing and Assembling Your Construct

Transgenic mice are generated as an easy way to reliably produce one or more gene products from a promoter of your choice. The promoter:gene(s) cassette inserts randomly into the mouse genome, and you screen for mice that

contain the insert

express the desired protein as appropriate and

do not contain too many copies of the insert.

It can happen that the cassette inserts into over 100 sites in the mouse genome, and each insertion has the potential to mutate/alter/knockout the normal regulation or transcription of genes at the insertion locus, so you want a mouse with few integration sites (ideally, only one insertion).

If you want ubiquitous, sustained expression of your transgene, many labs have been successful with the hybrid promoter consisting of the chicken beta-actin promoter, CMV enhancers, and a large synthetic intron (CAG), which together act as a robust transcriptional control module.

Preparing your DNA for Pronuclear Injection at MIT

One of the most critical steps in making transgenic mice is preparing the DNA for microinjection. Poorly prepared DNA can be toxic to the mouse eggs, and contaminants can clog the microinjection needle that typically has an inside diameter of 0.5 microns at the tip. Therefore, you should use one of the following protocols to purify your DNA:

1. Using the appropriate restriction enzymes, isolate the gene fragment, including all promotor elements necessary for transcription and removing the entire plasmid backbone. (This latter step is highly recommended... although transgenics have been made without removing plasmid sequences, there is literature on plasmid sequences adversely affecting transgene expression.)

2. Isolate the digested DNA on an agarose gel, making sure to run the gel long enough to cleanly separate bands which are close in size.

3. Purify the transgene fragment using one of the following methods:

Electroelution followed by purification over a DEAE minicolumn; (Schleicher & Schuell Elutip minicolumn, catalog #27370) or

Qiaquick gel extraction kit (for 70bp – 10 kb fragments, cat# 28704)

** Do not use Geneclean or similar procedures since residual beads will clog the microinjection pipettes and can be toxic to mouse eggs

4. Precipitate the DNA in ethanol. Wash several times in 70% ethanol and dry the pellet under a vacuum. Be thorough with washes and then with the drying because residual salts and /or ethanol are toxic to embryos (phenol chloroform extraction of the eluted fragment is not recommended).

5. Resuspend the DNA in a small volume of injection TE buffer (final concentration 10mM TRIS, 0.25mM EDTA purified by filtration and not autoclaving) and accurately deterimine the DNA concentration by flourimeter, OD or quantitation on an agarose gel using a known marker (i.e., Hind III lambda). Note: Regardless of how you determine DNA concentration, it is necessary to run your fragment on a gel to assess its DNA purity.

6. We need a minimum DNA concentration of 20- 50ng/microliter and between 30-50 microliters of DNA solution for each injection series. Ideally, we prefer 20ng/ul with a total volume of 50ul.